Fig. 6

Api5 regulates PDK1/Akt and ERK pathways through FGF2. A Lysates were collected from control and Api5 OE acinar cultures on days 4, 8, 12 and 16 and immunoblotted to study the expression of a number of proteins. Fold change in expression between control and Api5 OE were calculated for (B) Bim, (C) cleaved caspase 9, (D) pERK 1/2, (E) pMEK1, (F) FGF2, (G) pFGFR1 (Y653/654) (H) pAkt (T308), (I) cMYC, (J) pPDK1 (S241) and (K) KRas. The normalisation of expression was done with GAPDH for Bim, cleaved caspase 9, cMYC, and FGF2, while for pERK, pMEK, pFGFR1, pPDK1 and pAkt, it was with their respective total protein. (For pFGFR1 and FGFR1, samples from same experiment processed in 2 parallel gels as probing with pFGFR1 antibody affects binding of FGFR1 antibody). L Immunoblotting of various proteins involved in the plausible molecular pathway using lysates collected from day 7 MCF10CA1a control and Api5 KD spheroid cultures. Quantification of the fold change in expression of (M) pERK 1&2 normalised to total ERK2, (N) FGF2 normalised to GAPDH, (O) pFGFR1 normalised to total FGFR1, and (P) pAkt T308 normalised to total Akt. Data pooled from n > 5 independent experiments. Statistical analysis was performed using the paired t-test. *P < 0.05, **P < 0.01, ***P < 0.001and ****P < 0.0001. Data pooled from N ≥ 3 independent experiments. (Note: In 6A- pFGFR1 blot, day 4 samples are reprobed separately to overcome the masking effect, this data is added to Additional file 5B)